Neurotoxins with enhanced target specificity

ABSTRACT

Modified neurotoxins that contain protease cleavage sites susceptible uniquely to proteases present in certain tissues are described. The toxins can be selectively activated by proteases in muscle or selectively inactivated by proteases in blood.

TECHNICAL FIELD

The invention is directed to modified neurotoxins which are deactivatedin tissues where toxic activity is undesirable and neurotoxins that areactivated at desired targets. More specifically, the invention concernsinsertion of cleavage sites in non-critical regions of the toxins whichare susceptible to protease activity in non-target tissues and to singlechain forms which are activated by proteases in the target.

BACKGROUND ART

Structure and Function

Neurotoxins, such as those obtained from Clostridium botulinum andClostridium tetani, are highly potent and specific poisons of neuralcells. Both the single known tetanus toxin and the multiplicity of knownbotulinum toxins comprise, in their activated forms, two peptide acidchains coupled through a disulfide link: a light chain (LC) of about 50KDa and a heavy chain (HC) of about 100 KDa. The toxins are synthesizedin vivo as single chains, which are not toxic. However, the toxinbecomes active when the single chain is nicked in a post-translationalmodification to form the separate LC and HC (linked by S—S).

The tetanus and botulinum toxins have lethal doses in humans of between0.1 ng and 1 ng per kilogram of body weight. They function by inhibitingneurotransmitter release in affected neurons. The tetanus neurotoxin(TeNT) acts mainly in the central nervous system, while botulinumneurotoxin (BoNT) acts at the neuromuscular junction and othercholinergic synapses in the peripheral nervous system. Both types act byinhibiting acetylcholine release from the axon of the affected neuroninto the synapse, resulting in paralysis. The effect of intoxication onthe affected neuron is long-lasting and until recently has been thoughtto be irreversible.

Only one form of tetanus neurotoxin is known; seven differentimmunologically distinct forms of botulinum neurotoxins termed BoNT/Athrough BoNT/G are known. While all of these types are produced byisolates of C. botulinum, two other species, C. baratii and C. butyricumalso produce toxins similar to /F and /E, respectively.

Regardless of type, the molecular mechanism of intoxication appears tobe similar. First, the toxin binds to the presynaptic membrane of thetarget neuron through a specific interaction between the heavy chains(HC) and a cell surface receptor; the receptor is thought to bedifferent for each type of botulinum toxin and for TeNT. The carboxylterminus of the HC appears to be important for targeting of the toxin tothe cell surface.

In the second step, the toxin crosses the plasma membrane of thepoisoned cell, is engulfed by the cell through receptor-mediatedendocytosis, and an endosome containing the toxin is formed. The toxinthen escapes the endosome into the cytoplasm of the cell. The escape isthought to be mediated by a conformational change brought about by theacidic environment within the endosome which is effected by a protonpump that decreases intraendosomal pH. At a pH of about 5.5 or lower,the sequence at the amino terminus of the heavy chain triggers thisconformational change. The conformation shift exposes hydrophobicresidues which permits the toxin to embed itself in the endosomemembrane and then translocate into the cytosol.

Once in the cytosol, reduction of the disulfide bond joining the HC andLC takes place. The entire toxic activity of botulinum and tetanustoxins is contained in the LC; which is a zinc (Zn⁺⁺) endopeptidase thatselectively cleaves “SNARE” proteins essential for recognition anddocking of neurotransmitter-containing vesicles with the cytoplasmicsurface of the plasma membrane, and fusion of the vesicles with theplasma membrane.

The “SNARE” proteins are of several forms which have differentialresponses to the various forms of toxin. TeNT, BoNT/B BoNT/D, BoNT/F,and BoNT/G cause degradation of synaptobrevin (also calledvesicle-associated membrane protein (VAMP)), a synaptosomal membraneprotein. Most of the cytosolic domain of VAMP extending from the surfaceof the synaptic vesicle is removed as a result of any one of thesecleavage events. Each toxin (except TeNT and BoNT/B) specificallycleaves a different bond.

BoNT/A and /E selectively cleave the plasma membrane-associated proteinSNAP-25; this protein is predominantly bound to and present on thecytosolic surface of the plasma membrane. BoNT/C cleaves syntaxin, anintegral protein having most of its mass exposed to the cytosol.Syntaxin interacts with the calcium channels at presynaptic terminalactive zones.

Both TeNT and BoNT are taken up at the neuromuscular junction. BoNTremains within peripheral neurons, and blocks release of theneurotransmitter acetylcholine from these cells. TeNT enters vesiclesthat move in a retrograde manner along the axon to the soma, and isdischarged into the intersynaptic space between motor neurons and theinhibitory neurons of the spinal cord. At this point, TeNT bindsreceptors of the inhibitory neurons, is again internalized, and thelight chain enters the cytosol to block the release of the inhibitoryneurotransmitters 4-aminobutyric acid(GABA) and glycine from thesecells.

Pharmaceutical Applications

Dilute preparations of BoNT have been used since 1981 as therapeuticagents in the treatment of patients having various spastic conditions,including strabismus (misalignment of the eye), bephlarospasm(involuntary eyelid closure) and hemifacial spasm. See e.g., Borodic, etal., Pharmacology and Histology of the Therapeutic Application ofBotulinum Toxin in Therapy with Botulinum Toxin 119-157 (Jankovic J. &Hallett, eds. 1994), hereby incorporated by reference herein. The toxinpreparations are delivered specifically and locally to the site of theneurons to be effected. BoNT/A is the most potent of the BoNT's, and thebest characterized. Intramuscular injection of dilute preparations ofBoNT/A has also been used effectively to treat spastic conditions due tobrain injury, spinal cord injury, stroke, multiple sclerosis andcerebral palsy. The extent of paralysis depends on both the dose andvolume delivered to the target site.

Clearly, it is desirable to confine the activity of the administeredtoxin to the target site. A number of strategies have been adopted,including, besides direct injection, implantation of a capsule pump oradministration of a slow release gel. However, the success of suchattempts has been far from complete. Because of the diffusion of thetoxin from the site of administration, systemic problems, such asdifficulty in swallowing have occurred. The reality of these undesiredeffects has limited the level of dosage which can be administered. Forexample, subjects needing treatment in both arms or both legs generallycannot be administered the toxin in both affected limbs simultaneouslydue to the side effects. It would thus be desirable to provide a form ofthe toxins which inherently acts specifically at its target site. Thepresent invention provides such modified forms.

DISCLOSURE OF THE INVENTION

The invention provides botulism and tetanus toxins, including variantsand derivatives thereof, which contain protease target sites innon-critical regions such that specificity of toxicity with regard to aparticular target tissue is conferred. In general, the toxins are activeat the neuromuscular junction and thus, should be toxic in muscletissue; they may also be active at sites in the central nervous system.Systemic spread occurs mainly through the bloodstream, and it would thusbe desirable to inactivate the toxin as soon as any molecules enter thebloodstream. By suitable choice of cleavage sites, susceptible to bloodor muscle proteases respectively, the toxins can be provided in a formwhich will be inactivated in blood, activated in muscle, or both. Thecleavage site must be provided in a region of the toxin where itspresence does not disrupt the activity of the toxin, but where cleavageresults in activation or inactivation as the case may be.

Thus, in one aspect, the invention is directed to a modified botulism ortetanus toxin wherein the modification comprises the inclusion of acleavage site in a domain that must remain intact for activity and wherethe cleavage site is susceptible to cleavage by a protease that ispresent in effective levels only in a tissue where toxic activity isundesirable and where the cleavage site itself does not inactivate thetoxin. In another aspect, the invention is directed to a modifiedbotulism or tetanus toxin wherein the modification comprises a targetcleavage site for a protease such that an inactive form of the toxin isactivated. In this case, the protease specific for the cleavage sitewould be present in an effective amount only in tissues where toxicityis desired.

Still another aspect, the invention is directed to toxins which aremodified to contain cleavage sites of both types. Thus, such a toxinwould be specifically activated in muscle but deactivated once it wastransported into the blood stream.

In still another aspect, the invention is directed to recombinantmaterials encoding the modified toxins and to methods to produce them.The invention is also directed to methods to treat conditions benefitedby neurotoxin activity which comprises administering the neurotoxins ofthe invention or administering expression systems for their localizedproduction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of botulism A toxin (SEQ ID NO:1)purified from Clostridium botulinum strain Hall A where residues 437-438which are cleaved during post-translational modification are adoptedfrom the conserved sequence obtained by DNA sequences of strainsNCTC2916 and 62A.

FIG. 2 is a graph showing the stability of botulism toxin A in humanserum.

FIG. 3 is a graph showing the resistance of botulism toxin A to variousproteases.

MODES OF CARRYING OUT THE INVENTION

The botulism and tetanus toxins which are modified in order to obtainthe desirable properties associated with the invention include, inaddition to the naturally occurring forms, variants and derivatives ofthese forms. Such variants include, for example, chimeric forms of thesetoxins in which a portion of the heavy chain or light chain of BoNT/A isreplaced by the corresponding region of BoNT/E or BoNT/G or tetanustoxin. Various combinations can be envisioned. The toxin may have aheavy chain from one native toxin combined with light chain of another.In addition, variants may contain, in regions that are irrelevant toactivity, 1-5 substitutions, preferably by amino acids which are similarin character—i.e., conservative amino acid substitutions. The variantsmay also contain deletions of 1-5, preferably 1-3, more preferably 1-2amino acids in regions where the activity is substantially unaffected.Derivatives of these toxins include forms that may be lipidated,PEGylated, phosphorylated, or otherwise derivatized by additionalcovalently bonded groups, including the N-terminal acylated andC-terminal amidated forms. Thus, the toxins which are modified accordingto the present invention may include a range of variants and derivativesof the naturally occurring toxins. As used and defined herein, the terms“botulism toxin” and “tetanus toxin” refer not only to the nativetoxins, but also to the variants and derivatives as described above.

The invention is directed to modified forms of botulism or tetanus toxin(including variants and derivatives as defined above) which containprotease cleavage sites. In the aspect wherein the cleavage site willresult in inactivation of the toxin, the site must be present in aregion wherein the presence of the cleavage site itself does not destroytoxic activity. By “does not destroy toxic activity” is meant that thetoxic activity of the modified form is at least 10%, preferably 25%,more preferably 50%, more preferably 75% and most preferably at least90% of the unmodified form. Forms wherein the toxic activity is equal toor exceeds that of the toxin itself are also included. Such non-criticalregions can be determined experimentally by assessing the resultingtoxicity of the modified toxin using standard toxicity assays such asthat described by Zhou, L., et al., Biochemistry (1995) 34:15175-15181,which describes an in vitro assay for the ability of light chain tocleave recombinantly produced SNAP-25. Other suitable assays commonlypracticed include simple injection into mice to evaluate lethality.These assays are described, for example, by Maisey, E.A., et al., Eur J.Biochem. (1988) 177:683-691.

However, rational decisions about the location of such sites can bebased on the known conformation of the toxins. The crystal structure ofbotulism toxin type A is described, for example by Lacy, D. B., et al.,Nature Structural Biology (1998) 5:898-902. Based on the crystalstructure, and other data obtained with respect to botulism toxin typeA, certain features are apparent as will be described below. Because ofthe similarity of all of the botulism toxins and tetanus toxins, thedescription that is set forth below in detail with respect to type A isapplicable as well to the remaining toxin types and to tetanus toxins.The primary amino acid sequences are similar in all cases, and thefunctions and mechanisms of action are similar as well. Briefly, itappears that the light chain is globular, containing a number of exposedloops of minimal secondary structure; the heavy chain comprises twoglobular regions and a paired double helix. An extension of the lightchain comprising a “belt” circumscribes a portion of the complex.Suitable regions for locating a protease target site, wherein cleavageresults in inactivation would be found between the binding and transportdomains, between the two globules of the binding domain of the heavychain, in the non-catalytic regions of the light chain, and in the beltregion.

Other features of the protein are also known. Recent studies of theBoNT/A light chain have revealed certain features important for theactivity and specificity of the toxin towards its target substrate,SNAP-25. Thus, studies by Zhou, et al., Biochemistry 34:15175-15181(1995) have indicated that when the light chain amino acid residueHis₂₂₇ is substituted with tyrosine, the resulting polypeptide is unableto cleave SNAP-25; Kurazono, et al., J. Biol. Chem. 14721-14729 (1992)performed studies in the presynaptic cholinergic neurons of the buccalganglia of Aplysia californica using recombinant BoNT/A light chain thatindicated that the removal of 8 N-terminal or 32 C-terminal residues didnot abolish toxicity, but that removal of 10 N-terminal or 57 C-terminalresidues abolished toxicity in this system. Most recently, the crystalstructure of the entire BoNT/A holotoxin has been solved; the activesite is indicated as involving the participation of His₂₂₂, Glu₂₂₃,His₂₂₆, Glu₂₆₁, and Tyr₃₆₅. Lacy, et al., supra. (These residuescorrespond to His₂₂₃, Glu₂₂₄, His₂₂₇, Glu₂₆₂ and Tyr₃₆₆ of the BoNT/A Lchain of Kurazono et al., supra.) Interestingly, an alignment of BoNT/Athrough E and TeNT light chains reveals that every such chain invariablyhas these residues in positions analogous to BoNT/A. Kurazono, et al.,supra.

The catalytic domain of BoNT/A is very specific for the C-terminus ofSNAP-25 and appears to require a minimum of 17 SNAP-25 amino acids forcleavage to occur. The catalytic site resembles a pocket; when the lightchain is linked to the heavy chain via the disulfide bond between Cys₄₂₉and Cys₄₅₃, the translocation domain of the heavy chain appears to blockaccess to the catalytic pocket until the light chain gains entry to thecytosol. When the disulfide bond is then reduced, the catalytic pocketis “opened” and the light chain is fully active. As described above,VAMP and syntaxin are cleaved by BoNT/B, D, F, G and TeNT, and BoNT/C₁,respectively, while SNAP-25 is cleaved by BoNT/A and E.

While the presence of the cleavage site itself must permit the toxicactivity to be retained, actual cleavage at the site must result ininactivation. By inactivation in this context is meant that the toxinretains only 50% of the toxicity, preferably only 25% of the toxicity,more preferably only 10% of the toxicity, more preferably only 1% of thetoxicity of the uncleaved form. Thus, while the position of the siteshould be in a non-critical region with respect to the site itself,cleavage at that site must have a substantial effect.

For the design of a botulism toxin which can be inactivated by blood,protease sites which are recognized by proteases relatively uniquelyfound in the bloodstream are desirable. Among these proteases are thoseset forth below in Table 1 which also describes their recognition sites.

TABLE 1 Proteases Present in Blood Blood protease Substrate SpecificityThrombin P4-P3-P-R/K*P1'-P2'-P3/P4 hydrophobic; P1'/ P2' non-acidicP2-R/K*P1' P2 or P1' are G Coagulation Factor Xa I-E/D-G-R* CoagulationFactor XIa R* Coagulation Factor XIIa R* Coagulation Factor IXa R*Coagulation Factor VIIa R/K* Kallikrein R/K* Protein C R* MBP-associatedserine R* protease Oxytocinase N-terminal C* Lysine carboxypeptidaseC-terminal R/K* *indicates the peptide bond this protease will cleave.

As is clear, coagulation factors XIa, XIIa, IXa and VIIa as well askallikrein, protein C, MBP-associated serine protease, oxytocinase andlysine carboxypeptidase have relatively nonspecific target sites, whilecoagulation factors Xa and thrombin provide the opportunity for morespecificity.

In designing a thrombin or coagulation factor Xa site into a botulismtoxin, the location of the inserted site is, as described above, suchthat the presence of the site will not interfere with activity of thetoxin, but cleavage at the site will destroy or vastly inhibit theactivity of the toxin. In general, the early steps of the action can betargeted by placing the site into the receptor binding region or theinternalization region in the heavy chain, but away from the functionaldomains within these regions. Insertion sites in the heavy chainreceptor binding domain should be away from receptor binding grooves andin all cases the sites should be selected so as to be on the surface ofthe protein so that blood proteases can freely access them.

Table 2 indicates some examples of site modifications for cleavage bythrombin in the botulism toxin A amino acid sequence set forth inFIG. 1. In the table, the underline indicates a thrombin recognitionsite, the residues in bold are the additional amino acids; residueswhich were present in the native sequence but are eliminated when thesite is inserted are in parentheses. An asterisk indicates the peptidebond the thrombin will cleave.

TABLE 2 Thrombin Site Insertion Amino acid residue Location Example ofthrombin site insertion 930 -AIVYNS-935 (SEQ ID NO:2) H_(C) -A-I-R*G(VY)-N-S- (SEQ ID NO:3) -A-I-P-R* (VY)-N-S- (SEQ ID NO:4)-A-I-P-R*V-Y-N-S- (SEQ ID NO:5) 1060 -RDTH-1063 (SEQ ID NO:6) H_(C) -G-R*D-T-H- (SEQ ID NO:7) 1136 -KGPRGSVMT-1145 (SEQ ID NO:8) H_(C) - I(K)-G-P-R*G-S-V-M-T- (SEQ ID NO:9) 1165 -ASGNKDN-1171 (SEQ ID NO:10)H_(C) -A-S-G-G(N)-K*D-N- (SEQ ID NO:11) -A- L(S)-G-P (N)-K*G (D)-N- (SEQID NO:12)

The locations of the sites proposed are also set forth in FIG. 1.

Thus, for the inactivating cleavage, the protease should be one presentin high levels in blood. A suitable protease in this regard is thrombin,which, as shown below, occurs in blood in levels sufficient todeactivate the modified form of the toxins herein. By “effective” levelof the protease is meant a concentration which is able to inactivate atleast 50%, preferably 75%, more preferably 90% or greater of the toxinwhich enters the bloodstream at clinically suitable levels of dosage.

In general, the dosage levels for botulism toxins are on the order ofnanogram levels of concentration and thus are not expected to requirehigher concentrations of protease.

With respect to forms of toxins which are selectively activated bymuscle, preferably these comprise the single chain forms of the toxinprecursor. Such forms, which are resistant to proteolytic activity bythe Clostridium itself, have been designed for recombinant production inother organisms such as E. coli. Indeed, in some cases, the Clostridiumproduces mostly the uncleaved single chain form.

Single chain forms with a proteolytic site in the interchain loop regionfor cleavage by enzymes applied in vitro is described in application No.60/150,710 filed 25 Oct. 1999, and incorporated by reference. Asdescribed in this application, a single chain form of tetanus orbotulism toxin is constructed by ligating the nucleotide sequencesencoding light and heavy chain through a linker region corresponding,for example, to residue 437-448 of FIG. 1 (SEQ ID NO:1). In the case ofthe present invention, this linker region is provided with a target sitefor a protease which is specifically present at sufficientconcentrations in muscle to effect cleavage at this site.

As the production of the single chain form involves geneticmanipulation, (as does the construction of the form which is subject toinactivation in blood) mixing and matching of various regions of theseven botulism toxins and the tetanus toxin is well within the skill ofthe artisan. Thus, a botulism A light chain might be used in combinationwith botulism toxin B heavy chain or with a tetanus heavy chaintransmembrane region and a botulism G receptor recognition region. Theparticular toxin exemplified as a single chain product in theabove-referenced provisional application is a single chain tetanustoxin, but clearly other single chain toxins with appropriate proteasesites could be readily engineered. Thus, in the present invention, thenature of the protease cleavage site will be altered so as to besusceptible to muscle proteases.

The substrate neurotoxins which can be modified according to the methodof the invention are those natively occurring as described in theBackground section above, or can be themselves modified such as thosedescribed in PCT publications WO95/32738, WO96/33273, WO98/07864 andWO99/17806, all incorporated herein by reference. As stated above, theselectively activated and inactivated toxins of the invention have thefurther advantage that heavy chain and light chain components of theneurotoxins can be readily mixed and matched, and prepared as chimeras.

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLE 1 Thrombin Activity in Blood

The substrate for assay of thrombin proteolytic activity is theGST-SNAP-25 (a.a.128-197) fusion protein with a thrombin cleavage siteinserted between the two proteins. The substrate was added in a solutioncontaining 10 μg/1.55 μl to 8.45 μl of PBS or blood preparations to makethe final volume at 10 μl. The mixtures were incubated at 37° C. One μl(containing 1 μg of the substrate) used in each treatment was mixed with1 μl of 2× sample buffer with DTT and boiled for 5 minutes beforeloading to 18% SDS-PAGE gel. The gel was then blotted and probed with Ab197, which recognizes the C-terminal end of the cleaved or uncleavedSNAP-25₁₂₈-197.

Results showed that the substrate was cleaved by serum and at a muchlower level, by whole blood. No cleavage product was detected byincubating with plasma. The lack of thrombin activity in the plasmasuggests the majority of the enzyme is probably still in its proenzymeform (prothrombin). Prothrombin is activated to thrombin as a result ofa cascade of proteolytic enzymes that initiate blood coagulation, andthus thrombin is present in the serum because the clotting has beentriggered, but not in plasma where the clotting was prohibited by addingcitrate. the trace amount of thrombin cleavage product seen in thesample incubated with citrated whole blood indicates that a low level ofclotting pathway activation has occurred.

As typically these toxins are injected at very low (nanogram) levels,the thrombin levels in blood, even at the low levels of activation shownmay be sufficient to effect at least sufficient cleavage substantiallyto inactivate the toxin. In addition, the trauma caused by injection ofthe toxin may trigger activation of prothrombin to elevate these levels.

EXAMPLE 2 Stability of Native BoNT/A with Respect to Proteases

To test the stability of unmodified BoNT/A in human serum, BoNT/A wasadded at 10 μg/ml of human serum and incubated for the time specified.The activity of the toxin was measured by the absorbance at 450 nm inthe SNAP-25 assay described in co-pending application Ser. No.09/164,692, filed Aug. 25, 2000, claiming priority to provisionalapplication Ser. No. 60/150,710 filed Aug. 25, 1999, both incorporatedherein by reference. The absorbance value was compared at the dilutionof 1000 pg toxin per ml. As shown in FIG. 2, BoNT/A is fairly stable inhuman serum for up to 4 days and still retained 50% of the activityafter 5 days in the serum. Similar results were found when incubatingpure BoNT/A or complex with whole blood or plasma and the toxins werestable for up to 48 hours tested. These results suggest BoNT/A is stablein the blood without immediate breakdown by plasma protease andtherefore will potentially circulate to other muscles and organs andcause a systemic effect unless inactivated by the invention method.

The resistance of BoNT/A to various proteases. BoNT/A was incubatedovernight with excess purified restricted proteases at room temperatureand then the activity was analyzed by the SNAP-25 assay such as thatreferenced above. Factor Xa and thrombin are plasma proteases;enterokinase is present in the duodenal secretions into the GI tract.Activities are compared at 100 pg/ml dilution of each toxin reaction.The results are shown in FIG. 3. The toxin is resistant to theseproteases. Although the toxin incubated with thrombin lost ⅓ of itsactivity, it may not be very sensitive to thrombin degradation since anexcess of enzyme was used for a long period of time. SDS-PAGE gelanalysis of these treated toxins showed no cleavage.

1. A modified botulinum A neurotoxin comprising: a) a binding domaincomprising a first heavy chain portion able to interact with a surfacereceptor present in a target cell, wherein said first heavy chainportion comprises a mammalian blood protease cleavage site, saidmammalian blood protease cleavage site comprising a modification in oneor more regions selected from the group consisting of amino acids930-935 of SEQ ID NO: 1, amino acids 1060-1063 of SEQ ID NO: 1, aminoacids 1136-1144 of SEQ ID NO: 1 and amino acids 1165-1171 of SEQ ID NO:1; b) a translocation domain comprising a second heavy chain portionable to mediate the escape of a light chain portion of said neurotoxinfrom an endosome to the cytoplasm of said target cell; and c) anenzymatic domain comprising said light chain portion able to cleave aSNARE-protein present in said target cell; wherein said modifiedbotulinum A neurotoxin is able to interact with a surface receptor ofsaid target cell, able to mediate endosomal escape into the cytoplasm ofsaid target cell and able to cleave a SNARE protein present in saidtarget cell; and whereupon said modified botulinum A neurotoxin'sability to interact with a surface receptor of said target cell isinactivated upon cleavage of said mammalian blood protease cleavagesite.
 2. The modified botulinum A neurotoxin according to claim 1,wherein said mammalian blood protease cleavage site is cleaved by amammalian blood protease selected from the group consisting of Thrombin,Coagulation Factor VIIa, Coagulation Factor IXa, Coagulation Factor Xa,Coagulation Factor XIa, Coagulation Factor XIIa, Kallikrein, Protein Cand MBP-associated serine protease.
 3. The modified botulinum Aneurotoxin according to claim 1, wherein said mammalian blood proteasecleavage site is cleaved by Thrombin.
 4. The modified botulinum Aneurotoxin according to claim 1, wherein said mammalian blood proteasecleavage site is cleaved by Coagulation Factor VIIa.
 5. The modifiedbotulinum A neurotoxin according to claim 1, wherein said mammalianblood protease cleavage site is cleaved by Coagulation Factor IXa. 6.The modified botulinum A neurotoxin according to claim 1, wherein saidmammalian blood protease cleavage site is cleaved by Coagulation FactorXa.
 7. The modified botulinum A neurotoxin according to claim 1, whereinsaid mammalian blood protease cleavage site is cleaved by CoagulationFactor XIa.
 8. The modified botulinum A neurotoxin according to claim 1,wherein said mammalian blood protease cleavage site is cleaved byCoagulation Factor XIIa.
 9. The modified botulinum A neurotoxinaccording to claim 1, wherein said mammalian blood protease cleavagesite is cleaved by Kallikrein.
 10. The modified botulinum A neurotoxinaccording to claim 1, wherein said mammalian blood protease cleavagesite is cleaved by Protein C.
 11. The modified botulinum A neurotoxinaccording to claim 1, wherein said mammalian blood protease cleavagesite is cleaved by MBP-associated serine protease.
 12. The modifiedbotulinum A neurotoxin according to claim 1, wherein said modificationof amino acids 1060-1063 of SEQ ID NO: 1 is SEQ ID NO:
 7. 13. Themodified botulinum A neurotoxin according to claim 1, wherein saidmodification of amino acids 1136-1144 of SEQ ID NO: 1 is SEQ ID NO: 9.14. The modified botulinum A neurotoxin according to claim 1, whereinsaid modification of amino acids 1165-1171 of SEQ ID NO: 1 is selectedfrom the group consisting of, SEQ ID NO: 11 and SEQ ID NO:
 12. 15. Themodified botulinum A neurotoxin according to claim 1, wherein saidmodification of amino acids 930-935 of SEQ ID NO: 1 is selected from thegroup consisting of, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:
 5. 16.The modified botulinum A neurotoxin according to claim 15, wherein saidmodification of amino acids 930-935 of SEQ ID NO: 1 is selected from thegroup consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:
 5. 17. Themodified botulinum A neurotoxin according to claim 15, wherein saidmodification of amino acids 1060-1 063 of SEQ ID NO: 1 is SEQ ID NO: 7.18. The modified botulinum A neurotoxin according to claim 15, whereinsaid modification of amino acids 1136-1144 of SEQ ID NO: 1 is SEQ ID NO:9.
 19. The modified botulinum A neurotoxin according to claim 15,wherein said modification of amino acids 1165-1171 of SEQ ID NO: 1 isselected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12.20. A modified botulinum A neurotoxin comprising: a) a binding domaincomprising a first heavy chain portion able to interact with a surfacereceptor present in a target cell, wherein said first heavy chainportion comprises a mammalian blood protease cleavage site, saidmammalian blood protease cleavage site comprising a modification in oneor more regions selected from the group consisting of amino acids930-935 of SEQ ID NO: 1, amino acids 1060-1 063 of SEQ ID NO: 1, aminoacids 1136-1144 of SEQ ID NO: 1 and amino acids 1165-1171 of SEQ ID NO:1; b) a translocation domain comprising a second heavy chain portionable to mediate the escape of a light chain portion of said neurotoxinfrom an endosome to the cytoplasm of said target cell; and c) anenzymatic domain comprising said light chain portion able to cleave aSNARE-protein present in said target cell, wherein said light chainportion comprises a mammalian muscle protease cleavage site, saidmammalian muscle protease cleavage site comprising a modification inamino acids 430-452 of SEQ ID NO: 1; wherein said modified botulinum Aneurotoxin is able to interact with a surface receptor of said targetcell, able to mediate endosomal escape into the cytoplasm of said targetcell and able to cleave a SNARE protein present in said target cell; andwhereupon said modified botulinum A neurotoxin's ability to interactwith a surface receptor of said target cell is inactivated upon cleavageof said mammalian blood protease cleavage site; and whereupon saidmodified botulinum A neurotoxin's ability to cleave a SNARE proteinpresent in a target cell is activated upon cleavage of said mammalianmuscle protease cleavage site.
 21. The modified botulinum A neurotoxinaccording to claim 20, wherein said mammalian blood protease cleavagesite is cleaved by a mammalian blood protease selected from the groupconsisting of Thrombin, Coagulation Factor VIIa, Coagulation Factor IXa,Coagulation Factor Xa, Coagulation Factor XIa, Coagulation Factor XIIa,Kallikrein, Protein C and MBP-associated serine protease.
 22. Themodified botulinum A neurotoxin according to claim 20, wherein saidmammalian blood protease cleavage site is cleaved by Thrombin.
 23. Themodified botulinum A neurotoxin according to claim 20, wherein saidmammalian blood protease cleavage site is cleaved by Coagulation FactorVIIa.
 24. The modified botulinum A neurotoxin according to claim 20,wherein said mammalian blood protease cleavage site is cleaved byCoagulation Factor IXa.
 25. The modified botulinum A neurotoxinaccording to claim 20, wherein said mammalian blood protease cleavagesite is cleaved by Coagulation Factor Xa.
 26. The modified botulinum Aneurotoxin according to claim 20, wherein said mammalian blood proteasecleavage site is cleaved by Coagulation Factor XIa.
 27. The modifiedbotulinum A neurotoxin according to claim 20, wherein said mammalianblood protease cleavage site is cleaved by Coagulation Factor XIIa. 28.The modified botulinum A neurotoxin according to claim 20, wherein saidmammalian blood protease cleavage site is cleaved by Kallikrein.
 29. Themodified botulinum A neurotoxin according to claim 20, wherein saidmammalian blood protease cleavage site is cleaved by Protein C.
 30. Themodified botulinum A neurotoxin according to claim 20, wherein saidmammalian blood protease cleavage site is cleaved by MBP-associatedserine protease.
 31. The modified botulinum A neurotoxin according toclaim 20, wherein said modification of amino acids 930-935 of SEQ ID NO:1 is selected from the group consisting of, SEQ ID NO: 3, SEQ ID NO: 4and SEQ ID NO:
 5. 32. The modified botulinum A neurotoxin according toclaim 20, wherein said modification of amino acids 1060-1063 of SEQ IDNO: 1 is SEQ ID NO:
 7. 33. The modified botulinum A neurotoxin accordingto claim 20, wherein said modification of amino acids 1136-1144 of SEQID NO: 1 is SEQ ID NO:
 9. 34. The modified botulinum A neurotoxinaccording to claim 20, wherein said modification of amino acids1165-1171 of SEQ ID NO: 1 is selected from the group consisting of, SEQID NO: 11 and SEQ ID NO:
 12. 35. A modified botulinum A neurotoxincomprising a blood protease cleavage site, wherein said blood proteasecleavage site comprises a modification in one or more regions selectedfrom the group consisting of amino acids 930-935 of SEQ ID NO: 1, aminoacids 1060-1063 of SEQ ID NO: 1, amino acids 1136-1145 of SEQ ID NO: 1and amino acids 1165-1171 of SEQ ID NO:
 1. 36. The modified botulinum Aneurotoxin according to claim 35, wherein said mammalian blood proteasecleavage site is cleaved by a mammalian blood protease selected from thegroup consisting of Thrombin, Coagulation Factor VIIa, CoagulationFactor IXa, Coagulation Factor Xa, Coagulation Factor XIa, CoagulationFactor XIIa, Kallikrein, Protein C and MBP-associated serine protease.37. The modified botulinum A neurotoxin according to claim 35, whereinsaid mammalian blood protease cleavage site is cleaved by Thrombin. 38.The modified botulinum A neurotoxin according to claim 35, wherein saidmammalian blood protease cleavage site is cleaved by Coagulation FactorVIIa.
 39. The modified botulinum A neurotoxin according to claim 35,wherein said mammalian blood protease cleavage site is cleaved byCoagulation Factor IXa.
 40. The modified botulinum A neurotoxinaccording to claim 35, wherein said mammalian blood protease cleavagesite is cleaved by Coagulation Factor Xa.
 41. The modified botulinum Aneurotoxin according to claim 35, wherein said mammalian blood proteasecleavage site is cleaved by Coagulation Factor XIa.
 42. The modifiedbotulinum A neurotoxin according to claim 35, wherein said mammalianblood protease cleavage site is cleaved by Coagulation Factor XIIa. 43.The modified botulinum A neurotoxin according to claim 35, wherein saidmammalian blood protease cleavage site is cleaved by Kallikrein.
 44. Themodified botulinum A neurotoxin according to claim 35, wherein saidmammalian blood protease cleavage site is cleaved by Protein C.
 45. Themodified botulinum A neurotoxin according to claim 35, wherein saidmammalian blood protease cleavage site is cleaved by MBP-associatedserine protease.
 46. A modified botulinum A neurotoxin comprising ablood protease cleavage site and a muscle protease cleavage site,wherein said blood protease cleavage site comprises a modification inone or more regions selected from the group consisting of amino acids930-935 of SEQ ID NO: 1, amino acids 1060-1063 of SEQ ID NO: 1, aminoacids 1136-1144 of SEQ ID NO: 1 and amino acids 1165-1171 of SEQ ID NO:1; and wherein said muscle protease cleavage site comprises amodification in amino acids 430-452 of SEQ ID NO:
 1. 47. The modifiedbotulinum A neurotoxin according to claim 46, wherein said bloodprotease cleavage site is cleaved by a mammalian blood protease selectedfrom the group consisting of Thrombin, Coagulation Factor VIIa,Coagulation Factor IXa, Coagulation Factor Xa, Coagulation Factor XIa,Coagulation Factor XIIa, Kallikrein, Protein C, MBP-associated serineprotease, Ocytocinase and Lysine carboxypeptidase.
 48. The modifiedbotulinum A neurotoxin according to claim 46, wherein said bloodprotease cleavage site is cleaved by Thrombin.
 49. The modifiedbotulinum A neurotoxin according to claim 46, wherein said bloodprotease cleavage site is cleaved by Coagulation Factor Vlla.
 50. Themodified botulinum A neurotoxin according to claim 46, wherein saidblood protease cleavage site is cleaved by Coagulation Factor lXa. 51.The modified botulinum A neurotoxin according to claim 46, wherein saidblood protease cleavage site is cleaved by Coagulation Factor Xa. 52.The modified botulinum A neurotoxin according to claim 46, wherein saidblood protease cleavage site is cleaved by Coagulation Factor XIa. 53.The modified botulinum A neurotoxin according to claim 46, wherein saidblood protease cleavage site is cleaved by Coagulation Factor XIIa. 54.The modified botulinum A neurotoxin according to claim 46, wherein saidblood protease cleavage site is cleaved by Kallikrein.
 55. The modifiedbotulinum A neurotoxin according to claim 46, wherein said bloodprotease cleavage site is cleaved by Protein C.
 56. The modifiedbotulinum A neurotoxin according to claim 46, wherein said bloodprotease cleavage site is cleaved by MBP-associated serine protease. 57.The modified botulinum A neurotoxin according to claim 46, wherein saidblood protease cleavage site is cleaved by Ocytocinase.
 58. The modifiedbotulinum A neurotoxin according to claim 46, wherein said bloodprotease cleavage site is cleaved by Lysine carboxypeptidase.
 59. Themodified botulinum A neurotoxin according to claim 46, wherein saidmodification of amino acids 930-935 of SEQ ID NO: 1 is selected from thegroup consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO:
 5. 60. Themodified botulinum A neurotoxin according to claim 46, wherein saidmodification of amino acids 1060-1 063 of SEQ ID NO: 1 is SEQ ID NO: 7.61. The modified botulinum A neurotoxin according to claim 46, whereinsaid modification of amino acids 1136-1145 of SEQ ID NO: 1 is SEQ ID NO:9.
 62. The modified botulinum A neurotoxin according to claim 46,wherein said modification of amino acids 1165-1171 of SEQ ID NO: 1 isselected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12.